Over one hundred proteins important for cell growth, differentiation, and morphology, including many GTP-binding regulatory proteins (G proteins), require posttranslational modification by covalent attachment of an isoprenoid lipid (prenylation) for proper function (Tamanoi & Sigman, 2001). The three known enzymes that catalyze protein prenylation are the two Ca1a2X prenyltransferases, protein geranylgeranyltransferase type-I (GGTase-I) and protein farnesyltransferase (FTase), and a third enzyme, protein geranylgeranyltransferase type-II (RabGGTase; Casey & Seabra, 1996). GGTase-I modifies most monomeric G proteins in the Rho, Rac, and Rap subfamilies, and nine of the twelve heterotrimeric G protein γ subunits. Loss of GGTase-I function has dramatic biological effects, blocking the cell cycle at the G1 to S phase transition and promoting apoptosis (Li et al., 2002; Vogt et al., 1996). Since the demonstration that inhibition of FTase causes tumor regression in mice (Kohl et al., 1995), the prenyltransferase enzyme family has been studied in increasing detail. Drug design efforts have produced a number of Ca1a2X prenyltransferase inhibitors (PTIs) that are now in advanced clinical trials as anti-cancer treatments (Johnston, 2001).
Although the majority of these drug discovery efforts have focused on FTase inhibition, GGTase-I is increasingly of interest as a drug target. GGTase-I inhibitors (GTIs) have demonstrated efficacy in pre-clinical models of tumor progression (Sebti & Hamilton, 2000) and show promise in the treatment of smooth muscle hyperplasia (Stark et al., 1998). Recently, GGTase-I inhibitors were shown to attenuate clinical signs of disease in animal models of multiple sclerosis (Walters et al., 2002). GGTase-I has also been proposed as a target for countering parasitic infections such as malaria by selective inhibition of the parasite enzyme (Chakrabarti et al., 1998).
The use of prenyltransferasae inhibitors (PTIs) as human therapeutics has not been universally successful, however. Ongoing study has made clear that the use of protein prenyltransferase inhibitors (PTIs) as human therapeutics requires carefully calibrated levels of FTase or GGTase-I inhibition to avoid toxicity and unwanted side effects. The statin family of drugs blocks the committed step of cholesterol synthesis, in turn reducing farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) synthesis and ultimately reducing the protein prenylation levels in the cell. This chronic, low-level inhibition of protein prenylation has anti-cancer effects (Wong et al., 2002), but outright inhibition of all protein prenylation activity is toxic (deSolms et al., 2003). GTIs show anti-tumor activity (Sebti & Hamilton, 2000), and show promise in pre-clinical studies for the treatment of heart disease (Stark et al., 1998) and multiple-sclerosis (Walters et al., 2002). Reports of GTIs toxicity, however, are wide ranging, with side-effects ranging from benign (Sun et al., 1998) to lethal (Lobell et al., 2001). Overall, the clinical application of protein prenyltransferase to human disease would greatly benefit from a better understanding of inhibitor potency and selectivity.
Accordingly, it is an object of the presently disclosed subject matter to provide methods and compositions that can be used to identify new inhibitors of protein prenyltransferases. This object is achieved in whole or in part by the presently disclosed subject matter.
An object of the presently disclosed subject matter having been stated hereinabove, other objects will be evident as the description proceeds, when taken in connection with the accompanying Drawings and Examples as best described hereinbelow.